The present invention generally relates to an improved method and apparatus for the analysis of gas phase ions by ion mobility spectrometry and by mass spectrometry.
The present invention relates to methods for the analysis of samples by mass spectrometry and by ion mobility. The apparatus and methods for sample handling and analysis described herein are enhancements of the techniques referred to in the literature relating to mass spectrometry and ion mobility spectrometry—important tool in the analysis of a wide range of chemical compounds. Specifically, mass spectrometers can be used to determine the molecular weight of sample compounds. The analysis of samples by mass spectrometry consists of three main steps—formation of gas phase ions from sample material, mass analysis of the ions to separate the ions from one another according to ion mass, and detection of the ions. A variety of means and methods exist in the field of mass spectrometry to perform each of these three functions. The particular combination of the means and methods used in a given mass spectrometer determine the characteristics of that instrument.
To mass analyze ions, for example, one might use magnetic (B) or electrostatic (E) analysis, wherein ions passing through a magnetic or electrostatic field will follow a curved path. In a magnetic field, the curvature of the path will be indicative of the momentum-to-charge ratio of the ion. In an electrostatic field, the curvature of the path will be indicative of the energy-to-charge ratio of the ion. If magnetic and electrostatic analyzers are used consecutively, then both the momentum-to-charge and energy-to-charge ratios of the ions will be known and the mass of the ion will thereby be determined. Other well known mass analyzers are the quadrupole (Q), the ion cyclotron resonance (ICR), the time-of-flight (TOF), and the Paul ion trap analyzers. More recently, linear quadrupole ion traps [J. Schwartz, M. Senko, and J. Syka, J. Am. Soc. Mass Spectrom. 13, 659 (2002); J. Hager, Rapid Commun. Mass Spectrom. 16, 512 (2002)] have become more wide spread. And a new analyzer, the orbitrap, based on the Kingdon trap [K. Kingdon, Phys. Rev. 21, 408 (1923)] was recently described by A. Makarov [Q. Hu et al., J Mass Spectrom. 40, 430 (2005)]. The analyzer used in conjunction with the means and method described here may be any of a variety of these.
Before mass analysis can begin, gas phase ions must be formed from a sample material. If the sample material is sufficiently volatile, ions may be formed by electron ionization (EI) or chemical ionization (CI) of the gas phase sample molecules. Alternatively, for solid samples (e.g., semiconductors, or crystallized materials), ions can be formed by desorption and ionization of sample molecules by bombardment with high energy particles. Further, Secondary Ion Mass Spectrometry (SIMS), for example, uses keV ions to desorb and ionize sample material. In the SIMS process a large amount of energy is deposited in the analyte molecules, resulting in the fragmentation of fragile molecules. This fragmentation is undesirable in that information regarding the original composition of the sample (e.g., the molecular weight of sample molecules) will be lost.
For more labile, fragile molecules, other ionization methods now exist. The plasma desorption (PD) technique was introduced by Macfarlane et al. (R. D. Macfarlane, R. P. Skowronski, D. F. Torgerson, Biochem. Biophys. Res Commoun. 60 (1974) 616) (“McFarlane”). Macfarlane discovered that the impact of high energy (MeV) ions on a surface, like SIMS would cause desorption and ionization of small analyte molecules. However, unlike SIMS, the PD process also results in the desorption of larger, more labile species (e.g., insulin and other protein molecules).
Additionally, lasers have been used in a similar manner to induce desorption of biological or other labile molecules. See, for example, Cotter et al. (R. B. VanBreeman, M. Snow, R. J. Cotter, Int. J. Mass Spectrom. Ion Phys. 49 (1983) 35; Tabet, J. C.; Cotter, R. J., Tabet, J. C., Anal. Chem. 56 (1984) 1662; or R. J. Cotter, P. Demirev, I. Lys, J. K. Olthoff, J. K.; Lys, I.: Demirev, P.: Cotter et al., R. J., Anal. Instrument. 16 (1987) 93). Cotter modified a CVC 2000 time-of-flight mass spectrometer for infrared laser desorption of non-volatile biomolecules, using a Tachisto (Needham, Mass.) model 215G pulsed carbon dioxide laser. The plasma or laser desorption and ionization of labile molecules relies on the deposition of little or no energy in the analyte molecules of interest.
The use of lasers to desorb and ionize labile molecules intact was enhanced by the introduction of matrix assisted laser desorption ionization (MALDI) (K. Tanaka, H. Waki, Y. Ido, S. Akita, Y. Yoshida, T. Yoshica, Rapid Commun. Mass Spectrom. 2 (1988) 151 and M. Karas, F. Hillenkamp, Anal. Chem. 60 (1988) 2299). In the MALDI process, an analyte is dissolved in a solid, organic matrix. Laser light of a wavelength that is absorbed by the solid matrix but not by the analyte is used to excite the sample. Thus, the matrix is excited directly by the laser, and the excited matrix sublimes into the gas phase carrying with it the analyte molecules. The analyte molecules are then ionized by proton, electron, or cation transfer from the matrix molecules to the analyte molecules. This process (i.e., MALDI) is typically used in conjunction with time-of-flight mass spectrometry (TOFMS) and can be used to measure the molecular weights of proteins in excess of 100,000 Daltons.
Atmospheric Pressure Ionization (API) includes a number of ion production means and methods. Typically, analyte ions are produced from liquid solution at atmospheric pressure. One of the more widely used methods, known as electrospray ionization (ESI), was first suggested for use with mass spectrometry by Dole et al. (M. Dole, L. L. Mack, R. L. Hines, R. C. Mobley, L. D. Ferguson, M. B. Alice, J. Chem. Phys. 49, 2240, 1968). In the electrospray technique, analyte is dissolved in a liquid solution and sprayed from a needle. The spray is induced by the application of a potential difference between the needle and a counter electrode. The spray results in the formation of fine, charged droplets of solution containing analyte molecules. In the gas phase, the solvent evaporates leaving behind charged, gas phase, analyte ions. This method allows for very large ions to be formed. Ions as large as 1 MDa have been detected by ESI in conjunction with mass spectrometry (ESMS).
In addition to ESI, many other ion production methods might be used at atmospheric or elevated pressure. For example, MALDI has recently been adapted by Laiko et al. to work at atmospheric pressure (Victor Laiko and Alma Burlingame, “Atmospheric Pressure Matrix Assisted Laser Desorption”, U.S. Pat. No. 5,965,884, and Atmospheric Pressure Matrix Assisted Laser Desorption Ionization, poster #1121, 4th International Symposium on Mass Spectrometry in the Health and Life Sciences, San Francisco, Aug. 25-29, 1998) and by Standing et al. at elevated pressures (Time of Flight Mass Spectrometry of Biomolecules with Orthogonal Injection+Collisional Cooling, poster #1272, 4th International Symposium on Mass Spectrometry in the Health and Life Sciences, San Francisco, Aug. 25-29, 1998; and Orthogonal Injection TOFMS Anal. Chem. 71 (13), 452A (1999)). The benefit of adapting ion sources in this manner is that the ion optics (i.e., the electrode structure and operation) in the mass analyzer and mass spectral results obtained are largely independent of the ion production method used.
The elevated pressure MALDI source disclosed by Standing differs from what is disclosed by Laiko et al. Specifically, Laiko et al. disclose a source intended to operate at substantially atmospheric pressure. In contrast, the source disclosed by Standing et al. is intended to operate at a pressure of about 70 mtorr.
More recently, Takats et al. [Z. Takats, J. M. Wiseman, B. Gologan, and R. G. Cooks, Science 306, 471 (2004)] introduced yet another atmospheric pressure ionization method known as desorption electrospray ionization (DESI). According to Takats et al., DESI is a method for producing ions from analyte on a surface. Electrosprayed charged droplets and ion of solvent are directed at the surface under study. The impact of the charged droplets on the surface results in the desorption and ionization of the analyte to form gas phase analyte ions.
Analyte ions produced via an API method need to be transported from the ionization region through regions of differing pressures and ultimately to a mass analyzer for subsequent analysis (e.g., via time-of-flight mass spectrometry (TOFMS), Fourier transform mass spectrometry (FTMS), etc.). In some prior art sources, this was accomplished through use of a small orifice between the ionization region and the vacuum region. In other prior art, dielectric capillaries have been used to transmit ions entrained in a carrier gas from a high pressure ion production region into the vacuum chamber of mass spectrometers—see, for example, Fenn et al., U.S. Pat. No. 4,542,293 and Whitehouse et al., U.S. Pat. No. 5,844,237. In U.S. Pat. No. 6,777,672, incorporated herein by reference, Park describes a multiple section capillary for interfacing various ion production means and for transporting ions into the vacuum chamber of a mass spectrometer.
Importantly, ions are carried through the transfer capillary by entrainment in gas which is pumped from the ion production region, through the capillary, into the first vacuum region of the mass spectrometer. Typically, the gas pressure at the capillary inlet is about one atmosphere whereas the pressure at the capillary outlet, into the first pumping region, is between one and three millibar. Under these conditions, the velocity of the gas in the capillary is about 100 m/s. It is the “force” associated with this high velocity gas that is able to drive the ions away from the electrically attractive potential at the capillary entrance and towards the electrically repulsive potential at the capillary exit.
FIG. 1 depicts a prior art capillary 7 as incorporated in a prior art ion source. Capillary 7 extends from ion production region 40, into first vacuum region 35 of an ion source. O-ring 31 forms a seal between capillary 7 and the wall of vacuum region 35. Entrance end 26 of capillary 7 is substantially covered by apertured endcap electrode 33. Endcap 33 is composed of a chemically resistant, electrically conducting material such as stainless steel. When producing positive analyte ions, endcap 33 may be held at a potential of −4 kV and capillary entrance 26 may be held at a potential of −4.5 kV. Solution containing analyte is nebulized via sprayer 36. Sprayer 36 is held at near ground potential. As a result of the potential difference between sprayer 36 and endcap 33, the droplets formed via sprayer 36 are positively charged. Drying gas 27 is introduced into region 40 via the aperture in endcap 33. Solvent in the droplets formed via sprayer 36 evaporates into drying gas 27. Drying gas 27 may be heated to accelerate solvent evaporation. The complete, or near complete, evaporation of solvent from the analyte droplets results in gas phase analyte ions. The analyte ions are attracted by the electric field and by gas flow into entrance end 26 of capillary 7. Solvent is substantially removed from the flow of ions into entrance end 26 by the flow of drying gas 27 counter to the flow of the ions.
Once through capillary 7, the analyte ions are guided by a combination of gas flows and electric fields through differential pumping regions 65 and 67 to the outlet 69 of the ion source. On exiting the source through outlet 69, the ions either directly or indirectly enter the mass analyzer (not shown). In the mass analyzer the ions are mass analyzed and detected so as to yield a mass spectrum. Any known mass analyzer or combination of mass analyzers including time-of-flight, quadrupole, Paul trap, linear ion trap, orbitrap, electric or magnetic sector, or ion cyclotron resonance analyzers might be used.
One type of ion guide used in ion sources is the so called “ion funnel”. An ion funnel is disclosed by Smith et al. in U.S. Pat. No. 6,107,628, entitled “Method and Apparatus for Directing Ions and Other Charged Particles Generated at Near Atmospheric Pressures into a Region Under Vacuum”. One embodiment, illustrated in FIG. 2, consists of a plurality of elements, or rings 13, each element having an aperture, defined by the ring inner surface 20. At some location in the series of elements, each adjacent aperture has a smaller diameter than the previous aperture, the aggregate of the apertures thus forming a “funnel” shape, otherwise known as an ion funnel. The ion funnel thus has an entry, corresponding with the largest aperture 21, and an exit, corresponding with the smallest aperture 22. According to Smith et al., the rings 13 containing apertures 20 may be formed of any sufficiently conducting material. Preferably, the apertures are formed as a series of conducting rings, each ring having an aperture smaller than the aperture of the previous ring. Further, an RF voltage is applied to each of the successive elements so that the RF voltages of each successive element are 180 degrees out of phase with the adjacent element(s), although other relationships for the applied RF field would likely be appropriate. Under this embodiment, a DC electrical field is created using a power supply and a resistor chain to supply the desired and sufficient voltage to each element to create the desired net motion of ions through the funnel.
In co-pending application Ser. No. 11/219,639, incorporated herein by reference, the present inventor discloses a quadrupolar ion funnel having segmented electrodes. As detailed in the co-pending application by the present inventor, ring shaped electrodes are segmented into arcs as depicted in FIGS. 3A-C. According to the co-pending application, “ . . . FIG. 3B shows a cross-sectional view formed at line A-A in FIG. 3A. FIG. 3C shows a cross-sectional view formed at line B-B in FIG. 3A. In the preferred embodiment, segmented electrode 101 includes ring-shaped electrically insulating support 154 having aperture 169 through which ions may pass. Four separate electrically conducting elements 101a-101d are formed on support 154 by, for example, bonding metal foils to support 154. Importantly, elements 101a-101d cover the inner rim 169a of aperture 169 as well as the front and back surfaces of support 154 such that ions passing through aperture 169, will in no event encounter an electrically insulating surface. Notice also slots 151a-151d formed in support 154 between elements 101a-101d. Slots 151a-151d serve not only to separate elements 101a-101d but also removes insulating material of support 154 from the vicinity of ions passing through aperture 169. The diameter of aperture 169, the thickness of segmented electrode 101, and the width and depth of slots 151a-151d may all be varied for optimal performance. However, in this example, the diameter of aperture 169 is 26 mm, the thickness of electrode 101 is 1.6 mm, and the width and depth of slots 151 are 1.6 mm and 3.8 mm, respectively.
Ion mobility spectrometry (IMS) is a method whereby the “mobility” of analyte ions through a gas is measured under the influence of a static electric field. IMS is described in detail in the literature [see, for example, G. Eiceman and Z. Karpas, Ion Mobility Spectrometry (CRC. Boca Raton, Fla. 1994); and Plasma Chromatography, edited by T. W. Carr (Plenum, New York, 1984)]. At low electric field strengths—e.g. a few kilovolts per cm—the speed of analyte ions through a gas is measured. To start the measurement, ions are pulsed into the entrance of the mobility analyzer. In the mobility analyzer, a uniform electric field accelerates the ions towards the end of the analyzer. Collisions with gas in the analyzer tend to dampen the ion motion. The action of the electric field and collision of ions with the gas thus results in an average ion speed through the gas. At the far end of the analyzer, the ions strike a detector and are detected. By measuring the time between the introduction of ions into the analyzer and the detection of the ions, the speed of the ions, and therefore their mobility can be determined.
At low field strengths, the mobility of an ion is a constant relating the speed of the ion to the strength of the electric field. However, at high electric field strengths, the mobility of the ions varies with electric field strength. This gives rise to field asymmetric ion mobility spectrometry (FAIMS)—an extension of IMS which takes advantage of the change in ion mobility at high field strengths. FAIMS is described in detail in the literature [I. Buryakov, E. Krylov, E. Nazarov, and U. Rasulev, Int. J. Mass Spectrom. Ion Phys. 128. 143 (1993); D. Riegner, C. Harden, B. Carnahan, and S. Day, Proceedings of the 45th ASMS Conference on Mass Spectrometry and Allied Topics, Palm Springs, Calif., Jun. 1-4, 1997, p. 473; B. Carnahan, S. Day, V. Kouznetsov, M. Matyjaszczyk, and A. Tarassov, Proceedings of the 41st ISA Analysis Division Symposium, Framingham, Mass., Apr. 21-24, 1996, p. 85; and B. Carnahan and A. Tarassov, U.S. Pat. No. 5,420,424].
In recent years, IMS and FAIMS spectrometers have been combined with mass spectrometry. In U.S. Pat. No. 5,905,258 Clemmer and Reilly combine IMS with a time of flight mass spectrometer (TOFMS). This provides for a first analysis of the ions by IMS followed by a second analysis via TOFMS. Ultimately, this yields a two dimensional plot containing both the mobility and mass of the ions under investigation. The advantages of this type of combined analyzer over a mass spectrometer alone are described in detail in the literature [C. S. Srebalus et al., Anal. Chem. 71 (18), 3918 (1999); J. A. Taraszka et al., J. Proteom. Res. 4, 1223 (2005); R. L. Wong, E. R. Williams, A. E. Counterman, and D. Clemmer, J. Am. Soc. Mass Spectrom. 16, 1009 (2005)] and include the separation of chemical background from analyte species for an improved signal-to-noise ratio (S/N), and the separation of ions based on compound class or charge state for easier mass spectral interpretation.
Similarly, in U.S. Pat. No. 6,504,149, for example, Guevremont et al. combine a FAIMS device with a mass spectrometer. As detailed in the literature, a combined FAIMS mass spectrometer has similar advantages as an IMS mass spectrometer [A. Shvartsburg, K. Tang, R. Smith, J. Am. Soc. Mass Spectrom. 16, 2 (2005); D. A. Barnett, B. Ells, R. Guevremont, and R. W. Purves, J. Am. Soc. Mass Spectrom. 13, 1282 (2002)]. For example, a combined FAIMS mass spectrometer has an improved signal-to-noise ratio over a mass spectrometer alone because the FAIMS device can filter away the chemical background.
Several other methods of IMS separation have been demonstrated in the prior art. For example, J. Zeleny described a parallel flow ion mobility analyzer in “J. Zeleny, Philos. Mag. 46, 120 (1898).” In Zeleny's instrument, a voltage V is applied between two parallel grids separated by a distance h. Gas and ions flow through the grids parallel to the electric field. The electric field retards the motion of the ions such that the average velocity of ions between the grids is v=EK−u, where E is the electric field strength, K is the ion mobility and u is the air flow velocity. The mobility of the ions can be calculated as K=h/Et+u/E.
In U.S. Pat. No. 5,847,386, incorporated herein by reference, Thompson and Jolliffe suggest an ion mobility method wherein “ions are admitted into an RF multipole with an axial field, in the presence of cooling gas or drift gas, the ion velocity will reach a constant value which is proportional to the axial field. Ions of different size will drift at different velocities dependant on their shape, mass and charge, and be separated in time when they reach the exit of the device. If the exit gate . . . is opened at an appropriate time, only ions of a certain type will be admitted in the following analyzing device or other detector such as a mass spectrometer. This mobility separation may be applied to assist in the analysis of a mixture of ions . . . . ”
More recently Page et al. [J. S. Page et al., J. Mass Spectrom. 40, 1215 (2005)] and Laboda et al. [U.S. Pat. No. 6,630,662 incorporated herein by reference] employed the parallel flow analyzer method of Zeleny in combination with RF ion optical devices. However, the method of Page et al. results in a non-uniform gas flow—i.e. the flow direction and speed is dependent on both the axial and radial position within the device. Also the DC axial electric field is non-uniform and includes radial components. Furthermore, the RF confining field generated in the Page device includes an axial component. This tends to interfere with the mobility separation and introduces a mass effect to the separation. That is, the RF field has a greater effect on ions of a given mass range and a lesser effect on ions of another. Finally, in the method of Page et al. ions of a selected mobility cannot actually be isolated from ions of greater and lesser mobility. Rather, ions of high mobility are eliminated from a stream of ions having both high and low mobility. Similarly, Laboda does not teach a means and method whereby ions from a continuous ion source can be effectively introduced into an RF device for mobility analysis, how to generate a highly uniform gas flow or axial DC field, that an axial component of the RF field in the mobility analysis region should be avoided or how to eliminate this component of the field.
These shortcomings in prior art devices limit their mobility resolution, the sensitivity of the instruments employing them—i.e. many ions of interest are lost due to poor efficiency in the mobility device—and the accuracy of the mobility determinations. It is, in part, the purpose of the present invention to overcome these prior art limitations.